Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers
  • G01N33/57434—Specifically defined cancers of prostate
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
  • G01N2333/4701—Details
  • G01N2333/4718—Lipocortins

Definitions

• the present invention refers to novel antibodies recognizing native annexin A3. These antibodies are suitable for diagnostic and therapeutic applications.
• ANXA3 is significantly associated with the progression and the various stages of prostatic disease and prostate cancer. Still, little is known about the role and biology of ANXA3 in the human prostate. So far it has been shown that ANXA3 is part of so-called exosomes, small vesicles released form a variety of epithelia. There are indications that this phenomenon, which is the basis of the diagnostic performance of ANXA3, is due to a complex regulation of autoimmunity in prostate cancer.
• Protein biomarkers for therapy or diagnostics of prostate cancer and other epithelial cancers of urogenital tract are described in US 2005 130 876, WO 03 086 461 , WO 2005 078 124, EP 05 011 042.8 and EP 05 026 092.6. The content of these documents is herein incorporated by reference.
• ANXA3 has been known as marker for prostate cancer and specific antibodies for methods concerning differential diagnosis between benign, premalignant and malignant conditions in tissue sections are available, the measurement of ANXA3 in urine and réellee urine by these antibodies was met with a surprising difficulty.
• the conformation of "native" ANXA3 in urine is such that currently available antibodies, which are highly specific in Western blots or tissue staining, only detect a fraction of the protein. This is probably due to folding of ANXA3 in exosomes/prostasomes which results in non-accessibility of epitopes which are important for specific recognition of the protein.
• the aim of the present study was to investigate the quantitatively the abundance of native ANXA3 in a body fluid such as urine and réellee urine.
• the present invention refers to antibodies recognizing native annexin A3, particularly native human annexin A3. These antibodies are capable of specific binding to ANXA3 under non-denaturing conditions, i.e. in body fluids, such as urine and ceremoniese urine.
• the invention refers to an antibody recognizing native annexin A3, which is directed against an epitope in the amino acid sequence 59-67 of human annexin A3:
• a further preferred embodiment refers to an antibody recognizing native annexin A3, which is directed against a conformational epitope in the amino acid sequence 1-106 of human annexin A3, or an antigen-binding fragment of said antibody.
• this antibody may be directed against a conformational epitope in the amino acid sequence 1-34 of human annexin A3, or an antigen- binding fragment of said antibody.
• this antibody may be directed against a conformational epitope in the amino acid sequence 35-106 of human annexin A3, or an antigen-binding fragment of said antibody.
• Hybridoma cell lines producing antibodies TGC42 (DSM ACC 2972), TGC43 (DSM ACC 2970), TGC44 (DSM ACC 2976), TGC45 (DSM ACC 2974), TGC46 (DSM ACC 2975), TGC47 (DSM ACC 2977), TGC48 (DSM ACC 2971) and TGC49 (DSM ACC 2973) were deposited under the conditions of the Budapest Treaty at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH), Inhoffenstr. 7 B, 38124 Braunschweig, on 17 September 2008.
• the antibody of the invention may be a monoclonal, chimeric, humanized, human or recombinant antibody, e.g. a single chain antibody or an antigen- binding fragment of such an antibody, e.g. a proteolytic fragment or a recombinant single chain antibody fragment.
• the antibody is useful in medicine, particularly in human medicine. More particularly, the antibody may be used in diagnostic or therapeutic applications. Most preferably, the antibody is used for the diagnosis of cancer, such as prostate cancer.
• the antibody of the present invention is for use in a diagnostic assay which is performed under native conditions, e.g. a capture assay such as an ELISA wherein the presence and/or amount of native annexin A3 is determined.
• the antibody of the present invention may be conjugated to effector groups or labelling groups as known in the art.
• Effector groups may e.g. be selected from cytotoxic groups or compounds, e.g. chemotherapeutic agents or radionuclides.
• Labelling groups may be selected from any known labelling groups such as fluorescent groups, luminescent groups, enzyme labels, radioactive labels etc.
• the coupling of effector or labelling groups to antibodies may be carried out according to known techniques in the art.
• the present invention refers to a method for the diagnosis of cancer, preferably prostate cancer, wherein a body fluid sample is analyzed for the presence and/or amount of native annexin A3.
• the diagnostic method of the present invention involves a detection of native annexin A3, particularly native human annexin A3, by reaction with an antibody as described above.
• the sample is preferably a body fluid, such as urine orfuge urine.
• the cancer which may be diagnosed according to the present invention is preferably a cancer of the urogenital and/or gastro-intestinal tract, such as cancer of prostate, bladder, kidney, urethra, ovaria, uterus or colon.
• the cancer is an epithelial cancer.
• the cancer is prostate cancer.
• the invention comprises a differential diagnosis of a disease stage and/or a prognostic evaluation.
• a differential diagnosis of disease stages selected from: (i) a benign condition, particularly a benign prostatic condition such as benign prostatic hyperplasia (BPH), fibrosis and chronic prostatitis,
• a premalignant condition such as prostatic intraepithelial neoplasia of various stages (PIN-1-3) including high-grade prostatic intraepithelial neoplasia (HGPIN), and/or
• a malignant condition such as prostate cancer, particularly advanced prostate cancer in a progressed state indicated by Gleason rating.
• the invention allows a differential diagnosis between a benign or premalignant condition on the one hand and a malignant condition on the other hand.
• the presence, amount and/or distribution of native ANXA3 is determined in a sample, which may be a body fluid or a tissue sample. It should be noted that presence, amount and intracellular localization may be determined on a single tissue sample or on different samples, e.g. a body fluid sample and a tissue sample from the same subject.
• a high amount of native ANXA3 in a sample is primarily indicative for a benign condition.
• a moderate/low amount of native ANXA3, e.g. corresponding to weak/moderate staining of a tissue sample is indicative for a premalignant condition or to a early/early-intermediate stage malignant condition.
• the absence of native ANXA3 in a sample is indicative for a malignant condition, primarily for a malignant condition in a progressed state, e.g. in an intermediate or late stage.
• vesicles e.g. exosomes and/or prostatosomes
• vesicles can be obtained from urine or other body fluids by differential centrifugation, so the present invention relates to detection of native ANXA3 in any fraction of said body fluids.
• the method of the invention may encompass the determination of auto-antibodies against annexin A3.
• the presence of an autoimmune response against ANXA3, e.g. the presence of auto-antibodies against ANXA3, is primarily indicative for a malignant condition, particularly for a malignant condition in a progressed state.
• the present invention encompasses determination of polypeptides in a sample.
• this determination comprises immunological methods, wherein the presence, amount and/or localization of a component in a sample is determined using immunological test reagents.
• the reagent for determining native ANXA3 in a sample is preferably an antibody which is specific for native ANXA3, e.g. a polyclonal or a monoclonal antibody as described above.
• the reagent for determining ANXA3 auto-antibodies in a sample is preferably an ANXA3 polypeptide or a fragment thereof comprising specific epitopes important for recognition. More preferably, the ANXA3 polypeptide is a recombinant ANXA3 polypeptide, refolded to a native state, or native ANXA3 isolated from human or other mammalian sources.
• the method of the present invention may be carried out in any test format suitable for immunological determinations, including test formats suitable for automated devices.
• a reagent which carries a labelling group e.g. a visual marker, such as a latex or gold bead, a fluorescence marker group, an enzymatic marker group etc.
• Conjugates of reagents and labelling groups may be produced according to standard methods, e.g. by covalent coupling to reactive amino acid side groups of the reagent such as carboxy, amino and/or thiol groups with labelling groups, e.g. via bifunctional spacer molecules.
• the sample is preferably obtained from a human subject.
• the method is a non-invasive diagnostic procedure, wherein the sample may be e.g. a urine sample, particularly an urine sample, an imperativee urine sample or a faeces sample. If desired, the sample may be subjected to pretreatment procedures, e.g. gel filtration.
• the method may be a histochemical procedure wherein the sample may be a tissue sample, particularly a biopsy, e.g. a punch biopsy. In a histochemical procedure, a selective determination of intracellular or extracellular ANXA3 may be carried out by determining the localisation of ANXA3 within the sample.
• the invention comprises a semi-quantitative or quantitative determination of native ANXA3.
• This semi-quantitative or quantitative determination may involve evaluation of test results based on predetermined cut-off values and correlating the results of the evaluation with a disease stage. Cut-off values may be determined by determining native ANXA3 in samples from healthy persons and/or persons with a predetermined disease stage according to known methods. Further, the amount of native ANXA3 in a sample may also be determined by evaluation of stained tissue samples and correlating the results of the evaluation with a disease stage.
• the sample may be subjected to a fractionation procedure which allows separate determination of native ANXA3, e.g. in exosomes.
• the sample may be centrifuged in order to obtain a cell pellet and a supernatant whereby intracellular annexin A3 is determined in the cell pellet and extracellular annexin A3 is determined in the supernatant.
• the method comprises a selective determination of extracellular ANXA3.
• the method comprises a selective determination of intracellular ANXA3.
• the method of the present invention may additionally comprise the determination of further cancer markers, e.g. cancer markers.
• the determination of further markers may be carried out in the same sample where native ANXA3 is determined or in different samples, e.g. blood, serum and/or plasma samples.
• samples e.g. blood, serum and/or plasma samples.
• the determination of blood, serum or plasma markers in particular of at least one member of the kallikrein protease family, such as prostate specific antigen (PSA) and/or at least one epithelial cell marker, particularly prostate specific membrane antigen (PSMA).
• PSA prostate specific antigen
• PSMA prostate specific membrane antigen
• the present invention may be a screening procedure, wherein an individual or a group of individuals are tested for cancer, particularly prostate cancer.
• the method may also comprise a prognostic evaluation or therapeutic follow-up testing, wherein an individual who already has been diagnosed positive for cancer, particularly prostate cancer or a precursor stage thereof, is subjected to a prognostic evaluation and/or a therapy control monitoring.
• the invention relates to a prognostic evaluation of the disease progression, which is a valuable tool in any diagnostic assessment, particularly for therapy control.
• the prognostic evaluation may be based on determination of native ANXA3 alone or in combination with other markers such as PSA, PCA3 (Loeb S. Does PCA3 Help Identify Clinically Significant Prostate Cancer? Eur Urol. 2008 JuI 16.), PCADM-1 (Ohkia A, Hu Y, Wang M, Garcia FU, Stearns ME. Clin Cancer Res. 2004 Apr 1 ; 10(7):2452-8. Links Evidence for prostate cancer- associated diagnostic marker-1 : immunohistochemistry and in situ hybridization studies.), EPCA-2 (Katz MD, Kibel AS. Words of wisdom.
• EPCA-2 a highly specific serum marker for prostate cancer. Eur Urol. 2008 Jan; 53(1):210), (Diamandis EP. POINT: EPCA-2: a promising new serum biomarker for prostatic carcinoma? Clin Biochem. 2007 Dec; 40(18): 1437-9. Epub 2007 Sep 19.), (Leman ES, Cannon GW, Track BJ, Sokoll LJ, Chan DW, Mangold L, Partin AW, Getzenberg RH. EPCA-2: a highly specific serum marker for prostate cancer. Urology. 2007 Apr; 69(4):714-20), which are correlated positively to prostate cancer. For example, by histological evaluation and/or by measuring levels of PSA or further cancer markers, patients may be classified in a low risk group (e.g.
• PSA level ⁇ 10 ng/ml PSA level ⁇ 10 ng/ml
• an intermediate risk group e.g. PSA level > 10 and ⁇ 20 ng/ml
• a high risk group e.g. PSA level ⁇ 20 ng/ml
• Determination of ANXA3 in these patient groups may lead to further valuable information, particularly in patients having been classified as being in an intermediate risk group. If these intermediate risk patients are ANXA3 positive, the percentage of PSA-free survival is significantly higher than in patients having been determined as being negative for ANXA3.
• the invention allows a further risk stratification for individual patient groups.
• patients in a group having originally been classified as being in an intermediate risk group may be reclassified based on the results of the ANXA3 determination.
• Patients who are ANXA3 positive may be reclassified as being in a low risk group and patients who are ANXA3-negative (and optionally have an autoimmune response against ANXA3) are classified as being
• mice were immunized with recombinant human annexin A3. Thereby, monoclonal antibodies were generated which are capable of recognizing native annexin A3 in a capture ELISA combined with the polyclonal antiserum Petros (Wozny W, Schroer K 1 Schwall GP, Poznanovic S, Stegmann W, Dietz K, Rogatsch H, Schaefer G, Huebl H, Klocker H, Schrattenholz A, Cahill MA. Differential radioactive quantification of protein abundance ratios between benign and malignant prostate tissues: cancer association of annexin A3. Proteomics. 2007 Jan; 7(2):313-22).
• TGC43 DSM ACC2970
• TGC48 DSM ACC2971
• TGC42 DSM ACC2972
• TGC49 DSM ACC2973
• TGC45 DSM ACC2974
• TGC44 DSM ACC2976
• TGC47 DSM ACC2977
• all tested antibodies detect fragment vANA7 representing the N-terminal region of annexin 3 (cf. Figure 1 ).
• the binding sites of the tested antibodies thus, are within the N-terminal 106aa of annexin 3.
• the cell culture supernatants in a second set of experiments in the Western Blot were tested as to their reactivity to fragments vANA2 and vANA3 of annexin 3.
• vANA3 corresponds to fragment vANA2 except to the N-terminal region 34aa.
• the epitope of the monoclonal antibody 16C12 (TGC42), 18E9 (TGC43) and 14B10 (TGC49) is within the amino acid sequence EYQAAYGKE (positions 59-67 of annexin A3).
• the conformational epitope recognized by the antibodies 17D9 (TGC48) and/or 17F12 (TGC44) may be located within the amino acids 1-106, 1-34 and/or 35-106 of human annexin A3.
• Figure 3 shows epitopes recognized by ANXA3 specific antibodies in comparison to linear epitopes of the polyclonal anti-serum Petros. The figure shows a peptide scan of the polyclonal serum PETROS. The epitopes of different monoclonal antibodies are indicated.
• Figure 4 is a depiction of the ANXA3 spatial structure indicating epitopes recognized by antibodies recognizing denatured ANXA3 and antibodies recognizing native ANXA3 (present invention).

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• 2009
  • 2009-09-25 WO PCT/EP2009/062470 patent/WO2010034825A2/en active Application Filing
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